Mammals including humans need amplification by outer hair cells (OHCs) for their high sensitivity and sharp frequency selectivity. OHCs are also the most vulnerable cells in the inner ear and are involved in a majority of sensorineural hearing loss. Although OHC regeneration through either stem cell replacement or gene therapy provides an exciting option in the future, preventing hair cell loss is still the most direct and realistic approach to deal with hair cell loss, and including regenerated ones. Therefore, our proposal will focus on the maintenance of hair cells, particularly outer hair cells. OHC death is strongly connected with the generation of reactive oxygen species (ROS) that are known to destroy cells. Why OHCs, as compared to other cells in the organ of Corti, are more vulnerable to ROS is not fully understood. The main function of OHCs is to amplify mechanical signals through their somatic electromotility. This exclusive property is executed by a unique motor protein called prestin, which is expressed only in OHCs. In the past, studying OHC amplification mechanisms and preventing OHC loss were considered as two separate research fields. Unexpectedly, we discovered that non-functional point mutations in prestin (499-prestin mutant) leads to OHC death. In addition, prestin is not only associated with proteins involved in ROS generation, but prestin itself may have redox enzymatic activity, which could be a part of the OHC defense system to minimize ROS. These data indicate a close connection between prestin's function and the vulnerability of OHCs. The goal of this proposal is to understand this link, and subsequently to search for better strategies to prevent OHCs loss. Aim I of this proposal is designed to systematically characterize prestin's redox enzymatic activity in an in vitro system in order to reveal why the OHCs are the most vulnerable cells in the organ of Corti. This knowledge will allow us to develop a better strategy to prevent OHC loss. In Aim II, we will compare cell properties between wt-prestin and mutant 499-prestin expressing cells. Subsequent development of a system and method(s) to monitor the connection between prestin's function and cell vulnerability will then be used to test the effects of different anti-OHC loss reagents on prestin's functions. The information obtained from AIM II will help us to understand the relationship between prestin's function and cell death at the cellular level and provide guidance for selecting better approaches to protect OHCs. Aim III will focus on a search for new compounds through small molecule compound library screening procedures. Our purpose is to find novel compounds that may have specific and potent effects on prestin's redox enzymatic capacity, and are therefore usable for preventing OHC loss. Various cellular, biochemical and molecular biological methods will be used. Our strategies to prevent hair cell loss are based on knowledge derived from understanding prestin's function, which is unique to OHCs. This innovative approach will not only provide crucial information about basic mechanisms of cochlear amplification but it will also lead to a deeper understanding of processes associated with OHC death. The latter may lead to direct treatments or prevention of OHC damage. The proposed work is a first-step in translational research, which bridges basic mechanisms of cochlear physiology and treatments for hearing impairment. PUBLIC HEALTH RELEVANCE: Outer hair cells constitute the cochlear amplifier, essential for providing high sensitivity and frequency selectivity of hearing. They are also the most vulnerable cells in the inner ear and are involved in a majority of sensorineural hearing loss that affects the hearing of millions of people. Prestin, the motor protein of outer hair cells, is required for cochlear amplification. The goal of this proposal is to investigate prestin's protection role against oxidative stress in the ear. Data collected from these studies will not only expand knowledge of prestin as the OHC-based cochlear amplifier at the molecular level, but also produce a deeper understanding of mechanisms associated with outer hair cells loss, and may offer new methods for the prevention of OHC damage.